Rapid, point-of-care molecular diagnostics with Cas13

Shreeya Agrawal; Alison Fanton; Sita S Chandrasekaran; Bérénice Charrez; Arturo M Escajeda; Sungmin Son; Roger Mcintosh; Abdul Bhuiya; María Díaz de León Derby; Neil A Switz; Maxim Armstrong; Andrew R Harris; Noam Prywes; Maria Lukarska; Scott B Biering; Dylan C J Smock; Amanda Mok; Gavin J Knott; Qi Dang; Erik Van Dis; Eli Dugan; Shin Kim; Tina Y Liu; IGI Testing Consortium; Eva Harris; Sarah A Stanley; Liana F Lareau; Ming X Tan; Daniel A Fletcher; Jennifer A Doudna; David F Savage; Patrick D Hsu

doi:10.1101/2020.12.14.20247874

Rapid, point-of-care molecular diagnostics with Cas13

medRxiv [Preprint]. 2021 Apr 5:2020.12.14.20247874. doi: 10.1101/2020.12.14.20247874.

Authors

Shreeya Agrawal^{1

2}, Alison Fanton^{1

2

3}, Sita S Chandrasekaran^{1

2

3}, Bérénice Charrez³, Arturo M Escajeda⁴, Sungmin Son¹, Roger Mcintosh⁴, Abdul Bhuiya^{1

3}, María Díaz de León Derby^{1

3}, Neil A Switz⁵, Maxim Armstrong⁶, Andrew R Harris¹, Noam Prywes², Maria Lukarska^{2

7}, Scott B Biering⁸, Dylan C J Smock^{2

7}, Amanda Mok⁹, Gavin J Knott^{2

7

10}, Qi Dang², Erik Van Dis⁷, Eli Dugan^{2

7}, Shin Kim^{2

7}, Tina Y Liu^{2

7}; IGI Testing Consortium; Eva Harris⁸, Sarah A Stanley^{7

11}, Liana F Lareau^{1

2}, Ming X Tan⁴, Daniel A Fletcher¹, Jennifer A Doudna^{2

7

12

13

6

14}, David F Savage⁷, Patrick D Hsu^{1

2}

Affiliations

¹ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
² Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
³ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA.
⁴ Wainamics Inc., Pleasanton, CA, USA.
⁵ Department of Physics and Astronomy, San José State University, San José, CA, USA.
⁶ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
⁸ Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
⁹ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA.
¹⁰ Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
¹¹ School of Public Health, University of California, Berkeley, CA, USA.
¹² Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
¹³ Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
¹⁴ Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA.

Abstract

Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/μL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.

Publication types

Preprint

Abstract

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